The new CRISPR class expands its genetic engineering toolbox

Biomedical engineers at Duke University have used unexplored CRISPR technology to precisely regulate and edit human cell genomes.

With this new approach, researchers hope to significantly expand the CRISPR tools available to biomedical engineers, opening a new and diverse frontier for genome engineering technologies.

In a study published on 23 September Nature Biotechnology, Charles Gersbach, Associate Professor of Biomedical Engineering of the Rooney Family at Duke, and Adrian Oliver, Postdoctoral Researcher in the Gersbach Laboratory who led the project, describe how they have successfully operated Class 1 CRISPR systems to enable and disable the target genes. change the epigenome in human cells for the first time.

CRISPR-Cas is a defense system in which bacteria use RNA molecules and proteins associated with CRISPR (Cas) to target and destroy invasive virus DNA. The discovery of this phenomenon and the reuse of molecular machinery sparked a revolution in genome editing, as researchers learned how to use the tool to specifically target and modify DNA in human cells.

CRISPR-Cas9, the most widely used genome editing tool today, is classified in category 2 of the CRISPR system. Class 2 systems are less common in the bacterial world, but they are theoretically simpler to use because they depend only on a single protein case to target and cleave DNA.

Class 1 systems are not so simple, they rely on several proteins working together in a complex called Cascade (complex associated with CRISPR antiviral defense) to target DNA. After binding, Cascade recruits a Cas3 protein that cuts the DNA.

"If you were looking at individual CRISPR systems of all the bacteria in the world, nearly 90% of them are class 1 systems," said Gersbach. "CRISPR-Case biology is an incredible source for biotechnology tools, but until recently, everyone was interested only in a small piece of the cake."

To demonstrate the capabilities of the Class 1 system, Oliver linked gene enhancers to specific sites along an E cascade complex. Type I coli and targeted system to link gene promoters, which regulate gene expression levels. Because it did not include the Cas3 protein in the experiment, there was no DNA cleavage or change in the DNA sequence underlying. Experience has shown that the Cascade activator binds not only to the correct site and can increase target gene levels, but does so with a precision and specificity comparable to those of CRISPR / Cas9.

Oliver repeated the process using type I cascade complexes from an additional bacterial strain particularly effective for working on various target sites. It has also shown that the enhancer domain can be exchanged against a repressor to disable the target genes. Again, the researchers noted a precision and specificity comparable to the CRISPR / Cas9 methods.

"We found that Cascade's structure was remarkably modular, allowing a variety of sites to attach enhancers or repressors, which are excellent tools for altering gene expression in cells. "said Oliver. "Cascade's flexible nature makes it a promising genomic engineering technology."

Professors Rodolphe Barrangou and Chase Beisel, who are currently working at the Helmholtz Infection Research Center in Germany, encouraged Gersbach and Oliver to explore more complex Class 1 CRISPR systems. Barrangou is a microbiologist who has been studying the natural biology of various CRISPR defense mechanisms for nearly two decades. Beisel is a chemical engineer who worked with Barrangou on the development of microorganisms with CRISPR class 1 systems. They were both curious as to whether the Gersbach laboratory could use these systems in human cells, in the same way of their work with Cas9.

"This work and the resulting technologies are a fantastic example of how collaboration between disciplines from around the world and in North Carolina's research triangle universities can be extremely innovative and productive," says Barrangou, Distinguished Todd Professor. R. Klaenhammer in Probiotics Research at North Carolina State University. .

The team is now optimistic that their study and related work by other experts in the field will encourage new research on Class 1 CRISPR systems.

"The purpose of this project was to explore the diversity of CRISPR systems," said Gersbach. "There have been thousands of articles on CRISPR-Cas9 over the last decade, and yet we are constantly learning new ones.Through this study, we are applying this state of mind to the remaining 90% . "

Up to now, the team has shown that these class 1 systems are comparable to CRISPR-Cas9 in terms of accuracy and application. In thinking about future directions, they are curious about how these systems differ from their Class 2 counterparts and how these differences might be useful for biotechnology applications.

The team also wants to study how class 1 systems could solve the general problems of CRISPR-Cas research, particularly those that complicate potential therapeutic applications, such as immune responses to Cas proteins and simultaneous use of several types of CRISPR for different genome engineering functions.

"We know that CRISPR could have a significant impact on human health," said Gersbach. "But we are still at the beginning of understanding how CRISPR is going to be used, what it can do and what systems are available to us, and we hope this new tool will enable new areas of genome engineering. "